Purinergic P2X receptors (P2XRs) are a family of fast acting, cation-permeable ligand-gated ion channels (LGICs) activated by synaptically released extracellular adenosine 5'-triphosphate (ATP). Building evidence supports a role for P2XRs in the physiological and behavioral effects of alcohol (ethanol). However, there remains a paucity of knowledge regarding the molecular targets and actions of ethanol in P2XRs. The Specific Aim of my NRSA application addresses this issue by testing the hypothesis that positions 46, 331 and 336 within the ectodomain-TM interfaces of P2X4 receptors regulate the modulatory effects of ethanol in the neuronal environment. My hypothesis is focused on P2X4Rs because: 1) recent findings identified p2rx4 as one of the genes undergoing significant modulation in an alcohol paradigm;2) P2X4Rs are the most widely expressed P2XR subtype in the CNS ranging from neurons to glial cells;3) P2X4Rs show the highest sensitivity to the action of ethanol among all ethanol-sensitive P2XRs and 4) our laboratory recently found that these sites when mutated can reduce or alter the inhibitory effect of ethanol in P2X4Rs that are important for the action of ethanol without significantly altering receptor function (i.e., EC50, Imax, hill slope) when tested in Xenopus oocytes. A primary goal of our laboratory is the development of mutant P2XRs that are insensitive to ethanol. These mutant receptors can then be used to investigate the roles of specific P2XRs in the physiological and behavioral effects of ethanol through the development of mutant knock-in and null (knock- out) animals. The investigations that are proposed in this application represent a key step towards achieving this goal by determining if the findings by our laboratory in Xenopus oocytes translate to neurons. Importantly, the experiments that I will do over the course of my studies will provide me with significant training in molecular biology, lentiviral technologies, primary cell culture methods, pharmacology, and electrophysiological approaches through: 1) The development and use of a lentiviral vector based gene delivery system to express WT and mutant P2X4Rs in neurons and 2) Translation of the findings from an Xenopus oocyte model to a neuronal model using whole cell patch clamp electrophysiology. Completion of my training plan will significantly increase my knowledge and expertise regarding the role of P2XRs in the action of ethanol. In addition, completion of my investigations will represent a major step forward in my career development towards becoming an independent alcohol researcher in an academic setting. Importantly, results from the studies that I and my mentors have proposed in my NRSA application will be valuable in a larger context because so many people are affected by alcohol and this work will provide valuable information that, in the long term, may be used to guide the development of novel therapeutic compounds, targeting P2X receptors, which could be efficacious in the treatment of alcohol-related disorders. PUBLIC HEALTH RELEVANCE: Alcohol abuse and dependence are major problems that affect an estimated 14 million people, costs approximately 100,000 lives and nearly $200 billion annually in the United States. The search for better drugs to prevent and treat alcohol problems is impeded by our lack of knowledge regarding the initial molecular sites upon which alcohol acts in the brain that cause its behavioral effects. Results from the proposed studies should help fill this gap by providing key information regarding sites of action in a protein found in the brain that is believed to play a role in behavioral aspects of alcohol abuse and dependence.